Secretion of prolactin in the mammalian anterior pituitary gland is subject to continual modification by a variety of hormones and neurotransmitters. These modulators, in turn, often demonstrate multiple actions and/or actions on multiple cell types. The study of prolactin regulation is further complicated by the existence of subpopulations of prolactin-secreting cells which may exhibit differences in responsiveness to various modulators. The combined or sequential effects of these modulators on prolactin release are poorly understood. Furthermore, studies evaluating the individual effects of these factors have primarily utilized pituitary tissue or cell preparations without taking into account the functional heterogeneity among lactotrophs. Thus, definitive conclusions regarding the physiological roles of putative prolactin regulators remain elusive. In order to understand how regulators of prolactin secretion work in concert, it is first necessary to define how they individually. In addition, identification of subpopulations of prolactin secretors differentially affected by these regulators is inherent to this understanding. This proposal outlines a multidisciplinary experimental approach to the study of mechanism(s) of dopamine inhibition of prolactin release from individual lactotrophs dissociated from normal rat anterior pituitary tissue. Viable prolactin-secreting cells will be identified using the immunologically-based reverse hemolytic plaque assay. Prolactin secretors will be subgrouped according to 1) how much prolactin they release (large plaque- vs. small plaque-formers), OR 2) whether they release only prolactin or release both prolactin and growth hormone. These cells will then be used to study cellular and membrane responses to dopamine using the gigaseal patch clamp technique. The versatility of this technique will enable evaluation of the roles of intracellular messenger systems as well as membrane ionic mechanisms in the mediation of dopamine's action(s) on prolactin release. Characterization of the mechanisms underlying the action of individual modulators will establish the necessary basis for understanding integrated neuroendocrine function during physiological changes in prolactin secretion. In addition, this research will provide a framework in which to examine cellular and membrane properties and abnormalities associated with pathologies of hormone secretion. As such, these studies have a direct bearing on the regulation of fertility and reproduction and in the clinical management of hyperprolactinemia.